ANTIMICROBIAL AGENT FIELD OF THE INVENTION

The present invention relates to a new process for the synthesis of highly pure 4-bromothynnol, as well as its use as an antimicrobial agent. More in detail, the invention concerns a cheap, efficient and low environmental impact method for the preparation of highly pure 4-bromothymol, that can be easily obtained by the selective bromination of thymol on position 4. The purified 4-bromothymol prepared by this method showed an antimicrobial activity higher than that of thymol and therefore it is proposed as an option technically and economically favorable in all the cases where thymol is used at the moment.

BACKGROUND OF THE INVENTION

It is well known that phenol derivatives are widely exploited in several fields of application, for example as disinfectant. In particular, different phenol-based compounds are currently used as antioxidant and antibacterial agents in biomedical area. Thymol (or 2-isopropyl-5-methylphenol, or 3-hy- droxy-4-isopropyltoluene following the lUPAC nomenclature) belongs to this class of compounds and it is the main component of thyme essential oils. There is a high thymol content also in ethereal oils of several Labiate (such as oregano or monarda) and it confers a typical flavor to this kind of oils. It can also be obtained by synthetic pathway as for example by cumin aldehyde, by mefa-cresol and isopropyl alcohol, or by the para-cymene.

Thymol is endowed with antibacterial, antioxidant and antinflamma- tory activity and it is widely exploited as active ingredient in several personal care products, such as hair-washing products, intimate washes, mouth- washes, hands soaps, products for onychomycosis treatment, as well as in - -

surfaces disinfection products and as active component in bactericidal and antifungal and mold resistant paints. It has an antiseptic strength higher than that of phenol, but its very low solubility in water hampers a more diffuse application in the medical and surgical practice.

Moreover, it is well known that thymol acts also against varroasi, a disease caused by the mite Varroa destructor, which attacks the honey bees colonies leading to bees-death. It is currently supposed that it is the parasite with the more prominent economic impact on apiculture industry.

Also the bromo-phenols derivatives are widely used in the agrichemi- cal and pharmaceutical fields due to their intriguing antibacterial, antifungal, antiviral and anti-inflammatory properties. Moreover, these derivatives are considered as important synthetic intermediates for further transformations in organic chemistry.

At present 4-bromothymol is commercially available but it is little known and scarcely exploited, likely because of its high sales cost. This is mainly caused by the expensive procedures needed for its preparation either by synthetic method and by the natural source recovery. Moreover, the currently available 4-bromothymol is characterized by a low level of purity, thus it is unsuitable for biomedical purposes.

As known, common methods for the thymol bromination are based on molecular bromine, a corrosive and toxic reagent, and other reagents and solvents potentially harmful for the environment and toxic for living being. One of this methods is described in the Japanese patent JPS5835136 (A), by Fuji Zouki Seiyaku KK, filed in 1981 ; according to it, thymol is dis- solved in a non-aqueous solvent, such as benzene or chloroform, and is reacted with molecular bromine dissolved in the same solvent, preferably in the presence of a 3-5 fold molar excess of tetrahydrofuran with respect to thymol. According to what is reported, the main product of the bromination reaction is the 6-bromothymol derivative (according to the described struc- ture, bromine atom is on the para position with respect the hydroxyl group, - -

so it is usually named 4-bromothymol).

Taking into account the environmental impact of the classical bromin- ation reactions, alternative methods based on the catalytic activity of haloperoxidase enzymes (HalPO) mainly collected by seaweed and fungi have recently been investigated. It is known that these enzymes have fundamental role in the biosynthesis of brominated compounds, such as indoles, terpenes and phenols; in the presence of hydrogen peroxide they can oxidize halogen anion to different halogenating species, depending on reaction conditions.

A useful haloperoxidase involved in the synthesis of brominated compounds is the vanadium-dependent bromoperoxidase (VerPO) isolated from the brown seaweed Ascophyllum nodosum. The bromide oxidation in the presence of H2O2 catalyzed by VerPO has proved to be a sustainable method for the preparation of several brominated derivatives and this reac- tion has been successfully adopted for the bromination of several substrates. Among them, also the oxidative bromination of phenol and substituted phenols has been performed; this reaction usually leads to the selective bromination on para position with respect to the hydroxyl group, together with the formation of the orto, para-ό ibrominated derivatives, follow- ing the classical procedure of the electrophilic aromatic substitution (D. Wis- chang et al., vanadium-dependent bromoperoxidases from Ascophyllum nodosum in the synthesis of brominated phenols and pyrroles, Dalton Trans. 2013, 42, 1 1926-1 1940).

Some chemo-enzymatic techniques, based on the vanadium-de- pendent chloroperoxidase isolated from some particular molds as catalysts, have been suggested for the bromination of phenols such as thymol (Fer- nandez-Fueyo et al., Chemoenzymatic Halogenation of Phenols by using the Haloperoxidase from Curvularia inaequalis, Chem. Cat. Chem., 2015, 7, 4035-4038).

Although chemo-enzymatic techniques have been introduced with the aim to decrease the environmental impact of halogenation reactions, and in particular of oxidative bromination of phenol-based compounds such as thymol, it is important to underline that a catalytic reaction proceeding with a Vanadium-based catalyst reached a complete substrate conversion, while yields of the haloperoxidases-catalysed reaction resulted lower than 50%, mainly because of the non-stability of the biocatalyst in the presence of the oxidant (i.e. H2O2) (F. Sabuzi et al., Thymol Bromination - A Comparison between Enzymatic and Chemical Catalysis, Eur. J. Inorg. Chem., 2015, 3519-3525).

Considering again the environmental impact, it is noteworthy that the catalytic processes recently reported in the literature as alternative to the chemoenzymatic processes mentioned above, make use of organic solvents that are environmentally harmful, expensive and hard to find. In particular,

· Kaur et al. (Kaur, R. et al., Synthesis of halogenated derivatives of thymol and their antimicrobial activities, Med. Chem. Res. 2014, 23, 2212-2217) propose the conversion of thymol in monobromothymol and dibromothymol by reaction with /V-bromosuccinimide;

• Maruya et al. (Maruya, M.R. et al., Oxidative bromination of monoter- pene (thymol) using dioxidemolybdenum(VI) complexes of hydra- zones of 8-formyl-7-hydroxy-4-methylcoumarin, Polyhedron 2015, 96, 79-87) describe the synthesis of dioxidomolibdenum(IV) complex based catalysts, then used for the oxidative bromination of thymol in various solvents;

· Maruya et al. (Maruya, M.R. et al., Vanadium(V) complexes of a trip- odal ligand, their characterisation and biological implications, Dalton Trans. 2015, 44, 17736-17755) reported the synthesis of further vanadium^) complexes and their exploitation as catalysts precursors for the selective bromination of thymol in aqueous medium; in this case, the authors highlight that if no excess of H2O2 is used, the yield . .

of the para-monobrominated product reaches 93%, and no formation of the dibrominated derivative has been observed;

• Maruya et al, in the 2016 (Maruya, M.R. et al., Oxidoperoxidotung- sten(VI) and dioxidotungsten(VI) complexes catalyzed oxidative bro- mination of thymol in presence of H2O2-KBr-HCIO4, Inorg. Chim.

Acta 2016, 440, 172-180) describe other catalysts based on complexes of oxidoperoxidotungsten(IV) and dioxidotungsten(IV) that catalyze the oxidative bromination of thymol in acidic medium (HCIO4) with H2O2 (30%) in the presence of KBr; in the optimized reaction conditions, the products selectivity follows the order: 4-bromothymol > 2,4-dibromothymol > 2-bromothymol.

Actually, the reaction leading to the synthesis of the different bromo- thymol derivatives, such as 4-bromothymol, with an environmental friendly and non-toxic procedure, has been already published by a research group including the present inventors in 2015 (Sabuzi F., Churakova E., Galloni P., Wever R., Hollmann F., Floris B, Conte V, Thymol Bromination - A Comparison between Enzymatic and Chemical Catalysis, Eur. J. Inorg. Chem. 2015, 3519-3525). In such article the thymol bromination reaction has been carefully investigated in various conditions, looking for the more suitable ones, in order to obtain the higher amount of 4-bromothymol with respect to the other reaction products, namely 2,4-dibromothymol and 2-bromothymol.

The process of the proposed catalytic synthesis was experimentally performed in aqueous solution, with equimolar amount of thymol and potassium bromide and with a common vanadium catalyst, i.e. ammonium meta- vanadate (NH ₄ VO3). Then, hydrogen peroxide was added and the reaction mixture pH was adjusted to 1 , and kept under stirring at 30°C for 24 hours. The reaction proceeded by the following scheme: - -

Afterwards products isolation was conducted through a conventional purification procedure with chromatographic column purification using a mixture hexane:dichloromethane 3:1 v/v as eluent. Using such purification method, 4-bromothymol was isolated with a degree of purity, up to 96%. Such method, however, is not suitable for a preparative industrial process, because it implies the use of a large amount of organic solvents, that are generally harmful, toxic and special procedures for their disposal have to be applied.

Concerning the bromothymol antimicrobial features, it is worth mentioning that the previously cited Indian research group supervised by Ran- jeet Kaur studied in 2014 the antibacterial activity of 2-bromothymol (Kaur et al, loc. cit.), but the results obtained showed for such compound a negligible antibacterial action. Such preliminary result, particularly negative, probably discouraged the study of the antibacterial activity of other bromo- minated derivatives of thymol, such as 4-bromothymol.

SUMMARY OF THE INVENTION

The present invention purposes the aim to furnish a synthetic method for the 4-bromothymol production with a purity higher than 99%, and that is easy to operate and that provides a reaction in aqueous medium, with no aid of organic solvents (that are generally harmful and toxic), that does not require particular methods for product purification (thus avoiding classical chromatography procedures and/or distillation) and that can be carried out at room temperature, in order to avoid energy consumption. According to the invention, it has been found that the conventional purification step by column chromatography to obtain highly pure 4-bromo- thymol after the oxidative bromination reaction described in the previous paper by the research group of the inventors, (Sabuzi et al., loc. cit.) can be completely avoided. In fact, to isolate 4-bromothymol from the reaction mixture, it is enough to perform an easy cold crystallization by an aliphatic hydrocarbon (such as pentane). By this approach the desired product is easily obtained with a high purity grade (>99%) and, notably, through a quick, environmentally friendly and cheap procedure, considering the minimum amount of pentane needed.

Differently from how described in the mentioned publication, the purification of 4-bromothymol is carried out by cold crystallization: the oil obtained by the reaction is dissolved in the minimum amount of the aliphatic hydrocarbon and kept cold between -10°C and -30°C for several hours. This approach allows exclusively the crystallization of 4-bromothymol, that is the main reaction product with respect to 2-bromothymol. The residual solid is repeatedly crystalized with the same hydrocarbon solvent, up to 10 times, and it provides a white crystalline solid, whose purity (>99%) has been verified by gas-chromatography analysis.

Besides developing an easy and cheap method to obtain the para- monobrominated thymol derivative as main reaction product, the inventors noticed that such derivative, i.e. 4-bromothymol, is endowed with an antimicrobial activity that is not only much higher than the one given by the previous technique (Kaur et al, loc. cit.) for 2-bromothymol, but also that it is at least 15 times higher than that of thymol.

DETAILED DESCRIPTION OF THE INVENTION

According to an embodiment of the process developed in the present invention, following the synthetic method previously described by the same - -

authors, starting thymol is suspended with potassium bromide (KBr), preferably at an equimolar amount, in an aqueous solution containing a catalytic amount of ammonium metavanadate between 1 -10% (NH ₄ VO3), preferably about 5 % in moles with respect to thymol or bromide. Subsequently, hydro- gen peroxide is added (preferably 2 equivalents) and perchloric acid (HCIO4) is added to obtain pH between 0,5 and 2 (preferably 1 ). The system is stirred at a temperature between 20 and 60 °C (preferably 25°C) for 12- 48 hours (preferably 24 hours). Finally, reaction products are extracted with ethyl acetate and the organic phase is dried over sodium sulfate (Na2SO4), gravity filtered and solvent is evaporated by rotary evaporator. An orange- brown in color oil is obtained, which contains the unreacted thymol together with the reaction products. The reaction proceeds by the following pathway:

thymol 4-bromothymol 2-bromothymol

where 4-bromothymol is formed in 5-6 fold excess with respect to 2-bromothymol, while the formation of the dibrominated derivative (2,4-dibromothy- mol) is negligible because of the approximatively equimolar quantity of the starting reagents.

At this point, differently from what has been reported in the cited paper, the purification of 4-bromothymol is easily performed by cold crystallization: the obtained oil is dissolved in the minimum amount of an aliphatic hydrocarbon solvent (preferably hexane or pentane are used) and stored at a temperature between -10°C and -30°C (preferably -20°C) for 1 to 20 hours - -

(preferably 7 hours). By this way, exclusively 4-bromothynnol crystallization occurs, that is then let to settle and washed with the same hydrocarbon solvent. The crystallization step can be repeated 5-10 times to recover as much product as possible. A white and crystalline solid is obtained, whose purity (>99%) is checked by gas-chromatographic analysis. The complete characterization of the obtained compound can be performed through common laboratory techniques, such as GC-MS and ¹ H NMR, with CDC as solvent.

Therefore, a specific object of the present invention is a process for the preparation of highly pure 4-bromothynnol including the following operations:

a) suspending thymol and an alkaline metal or ammonium bromide in a molar ratio between 0.8:1 and 1 .2:1 in an aqueous solution containing a catalytic amount of a transition metal-based catalyst;

b) adding hydrogen peroxide in a molar amount about equal to the sum of molar amount of thymol and bromide, and a strong acid, in a sufficient amount to obtain a final reaction mixture pH between 0.5 and 2;

c) stirring the reaction mixture at a temperature between 20 and 60 °C for a period of time between 12 and 40 hours;

d) extracting the reaction products obtained by step c) with a non-toxic organic solvent, anhydrifying the organic phase over solid sodium sulfate, filtering and evaporating the solvent, thus achieving an orange-brown oil;

characterized in that such oil obtained by step d), is dissolved in an aliphatic hydrocarbon solvent, in a weight ratio oil/solvent of from 1 :0.5 to 1 :4, and left to stand at a temperature between -10°C and -30°C for a period of time between 1 and 20 hours, thereby obtaining the crystallization of 4-bro- motimol only, which is decanted and washed with such hydrocarbon solvent to give a crystalline white solid, consisting of highly pure 4-bromothymol. - -

Preferably, the starting reagents (thymol and bromide salt of an alkaline metal or ammonium) involved in step a) are present in the reaction mixture in equimolar amount.

According to some specific embodiments of the invention, the cata- lyst used for the oxidative bromination of thymol is a vanadium-based catalyst, preferably chosen among ammonium metavanadate (NH ₄ VO3), vanadyl acetylacetonate, (VO(CH ₃ COCHCOCH ₃ )2), vanadyl sulfate (VOSO4), and different synthetic vanadium based-complexes, such as those recently investigated by Maruya et al. (for example Maruya et al., Dalton Trans. 2015, loc. cit.). The use of catalysts based on other transition metals such as molybdenum or tungsten, would be possible, but it cannot be excluded that such catalysts may also result in different reactions. Preferably, the catalyst is ammonium metavanadate (NH ₄ VO3), and it is present in the reaction mixture in step a) in an amount equal to the 1 -10% in moles with respect to thymol or bromide, preferably 5%.

According to a specific embodiment of the invention, the bromide salt of alkali metal or ammonium is potassium bromide (KBr), but other bromides that do not result in side reactions, such as pH variations can be used, hence NaBr, NH ₄ Br and LiBr should not give troubles at pH 1 , while transi- tion metals bromide salts could lead to side reactions, so they would not be suitable.

In the described process, the strong acid, added in step b), is preferably perchloric acid (HCIO4), and it is added in the proper amount to obtain a final pH equal to approximatively 1 .

According to a specific embodiment of the invention, during the step c), the reaction mixture is kept under stirring at a temperature between 20 and 60 °C, preferably at 25 °C. The expected time for the reaction step is between 12 to 40 hours, preferably a period of 24 hours.

Preferably, the non-toxic organic solvent for the above described step d) is chosen among ethyl acetate, pentane, hexane, toluene, tert-butyl- - -

methylether, diethyl ether, and petroleum ether (40°-60° or 40°-70°). By a theoretical point of view, also chlorinated solvents, such as chloroform and dichloromethane, can fulfill the task, but it is preferable to avoid them because of their toxicity.

Turning now to the part of the process which represents the innovative component of the present invention, the aliphatic hydrocarbon solvent, in which the oil obtained in the step d) is dissolved, is preferably pentane, even if effective alternatives are represented by hexane, heptane, octane, cyclohexane and petroleum ether (40°-60° or 40°-70°).

According to a specific embodiment of the invention, the oil mixture obtained from the step d) is dissolved in the mentioned aliphatic hydrocarbon solvent and it is kept at a temperature of about -20 °C for a period of 7- 8 hours. At the end of this period, the crystalline white solid, obtained by the crystallization is crystallized again, three times or more (up to 10 times) with the aliphatic hydrocarbon solvent previously used, and the resulting product is constituted by highly pure 4-bromothymol, as confirmed by GC, GC-MS and ¹ H-NMR in CDC analyses, as reported in the following Example 1 .

According to a further aspect, the object of this invention is the application of the product obtained by the above mentioned method as an anti- microbial agent, alternative to thymol, being considerably more effective than thymol, as observed by the trial runs under this invention and subsequently reported.

More in detail, a further specific subject of the invention is the exploitation of the purified 4-bromothymol as antimicrobial agent and/or disinfect- ant in products for the personal hygiene and inside bactericidal formulations and surfaces disinfectants.

Moreover, a further object of the present invention is the exploitation of the purified 4-bromothymol as antimicrobial, antibacterial, antifungal, antiparasitic agent and/or disinfectant in medicine. - -

DESCRIPTION OF THE DRAWINGS

Figure 1

It reports the Mass Spectrum of the solid white crystalline product obtained by the process described in Example 1 . This analysis together with the ¹ H- NMR confirmed that such product is 4-bromothymol.

Figure 2

It reports the ¹ H-NMR (using CDC as solvent) of the solid white crystalline product obtained by the process described in Example 1 . This analysis to- gether with the MS confirmed that such product is 4-bromothymol .

EXAMPLES

Some specific embodiments of the method of this invention are listed below to be kept as an example, but not limited to, together with the results of the experiments ran by using the final product.

EXAMPLE 1

5 mmoles of thymol (750 mg) and 5 mmoles of KBr (600 mg) were dissolved in 50 ml of an aqueous solution of NH ₄ VO3 5 mM, previously prepared dissolving 30 mg of NH ₄ VO3 (0,25 mmoles) in water.

Subsequently the following reagents were added, in the following order: 1 ml of H2O2 10,4 M (10 mmoles) and 430 μΙ of perchloric acid (65%) to reach a pH equal to 1 . The mixture was kept under stirring at 25°C, for 24 hours.

The reaction products were extracted with 3 portions of 50 ml of ethyl acetate and the organic phase dried over Na2SO4 and filtered. Solvent was evaporated under reduced pressure by a rotary evaporator.

The oil so obtained was dissolved in 1 ml of pentane and kept at - 20°C for 7 hours. A crystalline white solid was obtained, which was crystal- ized 5 more times in pentane with the same procedure. 800 mg of product with a purity >99% was obtained, in 70 % yield. - -

GC-MS and ¹ H-NMR (CDC as solvent) analyses of the solid, shown respectively in Figures 1 and 2 here attached, confirmed that the crystallized product is 4-bromothymol.

EXAMPLE 2

For a possible an industrial scale-up of the preparation of 4-bromothymol according to the invention, the following procedure was applied.

In a 200 L reactor, (preferably made in PVDF = polyvinyl idene fluoride) 1 .5 kg of thymol (10 moles) and 1 .2 kg of KBr (10 moles) were manually introduced. These reagents were dissolved in 100 liters of an aqueous so- lution of NH ₄ VO3 5 mM, prepared by dissolving 60 g of NH ₄ VO3 (0,5 moles) in water.

Subsequently the following reagents were added, in the following order: 2 liters of H2O2 10,4 M (20 moles) and 850 ml of perchloric acid (65%) to reach a pH equal to 1 . The system was stirred by a magnetic stirrer (pref- erably made by Sandvik Austenite Ferrite SAF2205 material), at 25°C for 24 hours.

The reaction products were extracted with 3 portions of 50 liters of ethyl acetate; the organic phase was initially dried over Na2SO4 and then filtered. Solvent was evaporated under reduced pressure.

An oil was obtained and it was dissolved in 2 L of pentane and kept at -20°C for 7 hours. A crystalline white solid was obtained, that is crystallized 5 times more by pentane to obtain a purity >99%. 1 ,6 kg of product were obtained about (70 % yield).

COMPARISON EXAMPLE 1 (Preparation of Bromothymol following Sabuzi et al.)

The method of preparation of 4-bromthymol following the procedure described in the article Sabuzi et al. (Sabuzi F., Churakova E., Galloni P., Wever R., Hollmann F., Floris B, Conte V, Thymol Bromination - A Comparison between Enzymatic and Chemical Catalysis, Eur. J. Inorg. Chem. 2015, 3519-3525) was repeated. - -

In particular, 5 mmoles of thymol (750 mg) and 5 mmoles of KBr (600 mg) were dissolved in 50 ml of 5 mM aqueous solution of NH ₄ VO3, previously prepared dissolving 30 mg of NH ₄ VO3 (0,25 mmoles) in water.

Subsequently the following reagents were added, in the following or- der: 1 ml of H2O2 10,4 M (10 mmoles) and 430 μΙ of perchloric acid (65%) to reach a final pH equal to 1 . The mixture was stirred at 25°C, for 24 hours.

The reaction products were extracted with 3 portions of ethyl acetate (50 ml_ each) and the organic phase dried over Na2SO4 and filtered. Solvent was removed under reduced pressure by a rotary evaporator.

Residual oil was purified by column chromatography using S1O2 and hexane/dichloromethane 3/1 v/v obtaining as a third fraction 4-bromothy- mol, as a crystalline white solid with a purity grade of 90-96%.

EXAMPLE 3 (Antimicrobial activity)

The performed tests highlight a remarkable antimicrobial activity of 4-bromothymol against several bacterial strains, such as Staphylococcus aureus, Streptococcus mutans, Acinetobacter baumannii, Enterococcus faecalis, Bacillus subtilis, Enterobacter sakazakii and Escherichia coli. It is known that such bacteria represent the main responsible of some common human infections.

The bacterial strains were plated for insulation on breeding ground

Tryptic Soy Agar (Liofilchem®, Italy). Starting from the insulation, fresh bacterial cultures were set up and kept grew up overnight in Tryptic Soy Broth (Liofilchem®, Italy) at the appropriate temperature conditions (preferably at 21 "C or 37 °C).

From each culture broth a portion was withdrawn and then re-suspended in sterile physiological solution with a 0,5 McFarland turbidity. By repeated dilutions, a final bacterial count equal to 1 10 ⁶ CFU/ml was obtained.

For the MIC determination (Minimum Inhibitory Concentration) the microdilution method on agar culture was used. - -

Different solutions of thymol, 2-bromothymol and 4-bromothymol in

DMSO were prepared having a concentration of 90 mg/ml. Each compound was diluted in serial mode and added to the culture medium Mueller-Hinton

Agar (Liofilchem®, Italy) to have a range of concentrations of the compound to be tested. Subsequently, bacterial suspensions were inoculated as spots

(10 μΙ) directly on agar and the cultures were incubated in aerobiosis at the suitable temperature conditions for 24 hours.

The Minimum Inhibitory Concentration for each strain was recorded as the lowest compound concentration, by which no bacteria growth can be detected on plate after a 24 hours incubation.

Each protocol for MIC determination was repeated at least 2 times, for each compound at each concentration.

Tests were performed on a wide range of bacterial strains and the activity of the compound of interest, namely 4-bromothymol, was compared with the one of thymol, an antimicrobial agent widely used in several industrial products, and with the one of 2-bromothymol, the main byproduct of the synthesis of the desired compound. In Table 1 are reported just a few of the obtained results, the ones considered most remarkable. Table 1

Minimum Inhibitory Concentration of thymol, 2-bromothymol and 4-bromothymol on different bacterial strains.

MIC (Mg/mL)

Species Strains

2-Bromothy- 4-Bromothy-

Thymol

mol mol

Staphylococcus 450 250 28

P.37 70798150 ^*

aureus

Staphylococcus 450 Resistant 28

P.31 70783698 ^*

aureus - -

on a reaction performed in water, with no organic solvents and with no complicated procedures for the purification of the final product, that is obtained in high yield and purity. Moreover, the reaction is performed at room temperature, so avoiding energy wasting usually due to the high reaction temperatures.

In conclusion, the process for 4-bromothymol production according to this invention, allows to reach three important advantages:

^■ Placing on market of 4-bromothymol (with a purity higher than 99%), as antimicrobial agent: in particular, due to its high cost and not acceptable purity of the product currently available, the antimicrobial features of 4-bromothymol were not previously investigated;

^■ Replacing thymol and 4-chlorothymol as antimicrobials: 4-bromothymol resulted up to 15 times more effective than the above mentioned - -

compounds.

The efficiency of the product antimicrobial action, the cheapness and the sustainability of the manufacturing process, represent the key aspects for the success of this product, as disinfectant agent and, in particular, as an antimicrobial.

The present invention has been described in relation to some forms of its specific implementation, but it is understood that some variations or changes could be applied by qualified specialists without for this reason departing from the relative scope of protection.